Paper

A dynamic intermediate state limits the folding rate of a discontinuous two-domain protein

Lamb, Don C, Barth, Anders, Agam, Ganesh

2022

Overview

Protein folding is an indispensable process for the majority of proteins after their synthesis from ribosomes in the cell. Most in vitro protein folding studies have focused on single-domain proteins. Hence, it is important to understand the folding process of multi-domain proteins, especially when domains are discontinuous. We choose the Maltose binding protein (MBP) as a model system. In particular, we studied a mutant of MBP that folds slowly. Here, using two- and three-color single-molecule Foerster resonance energy transfer (smFRET) experiments, we study the refolding of both the domains and the interaction between the domains of DM-MBP. Initial two-color smFRET measurements of the N-terminal domain (NTD) reveal the presence of a folding intermediate. The same folding intermediate is observed in measurements monitoring the C- terminal domain (CTD) and the NTD-CTD (N-C) interface. The refolding intermediate is dynamic on the sub-millisecond timescale. Quantitative analysis on underlying dynamic interconversions revealed a delay in NTD folding imposed by the entropic barrier being the primary cause for slow DM-MBP folding. Moreover, CTD folds after NTD completes the folding. Using three-color smFRET, we could show the NTD folds first and CTD later in a same protein. Molecular dynamic simulations for temperature-induced unfolding on WT- and DM-MBP identify a folding nucleus in the NTD, which is rich in hydrophobic residues, and explains why the two mutations slow down the folding kinetics. In the presence of the bacterial Hsp60 chaperonin system GroEL/ES, we observe that DM-MBP is dynamic within the chaperonin cavity but the chaperonin limits the conformational space of substrate. Hence, confinement aids DM-MBP in overcoming the entropic barrier. The study reports on the subtle tuning and co-dependency for protein folding between two-domains with a discontinuous arrangement.

• Publisher: Cornell University Library, arXiv.org
• Subject: Color / Energy transfer / Folding / Maltose / Molecular dynamics / Mutation / Protein folding / Proteins / Substrates